PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42

Formation of the apical surface and lumen is a fundamental, yet poorly understood, step in epithelial organ development. We show that PTEN localizes to the apical plasma membrane during epithelial morphogenesis to mediate the enrichment of PtdIns(4,5)P2 at this domain during cyst development in three-dimensional culture. Ectopic PtdIns(4,5)P2 at the basolateral surface causes apical proteins to relocalize to the basolateral surface. Annexin 2 (Anx2) binds PtdIns(4,5)P2 and is recruited to the apical surface. Anx2 binds Cdc42, recruiting it to the apical surface. Cdc42 recruits aPKC to the apical surface. Loss of function of PTEN, Anx2, Cdc42, or aPKC prevents normal development of the apical surface and lumen. We conclude that the mechanism of PTEN, PtdIns(4,5)P2, Anx2, Cdc42, and aPKC controls apical plasma membrane and lumen formation.     

SNX9 activities are regulated by multiple phosphoinositides through both PX and BAR domains

Sorting nexin 9 (SNX9) functions at the interface between membrane remodeling and the actin cytoskeleton. In particular, SNX9 links membrane binding to potentiation of N-WASP and dynamin GTPase activities. SNX9 is one of a growing number of proteins that contain two lipid-binding domains, a phox homology (PX) and a Bin1/Amphiphysin/RVS167 (BAR) domain, and localizes to diverse membranes that are enriched in different phosphoinositides. Here, we investigate the mechanism by which SNX9 functions at these varied membrane environments. We show that SNX9 has low-lipid-binding affinity and harnesses a broad range of phosphoinositides to synergistically enhance both dynamin and N-WASP activities. We introduced point mutations in either the PX domain, BAR domain or both that are predicted to disrupt their functions and examined their respective roles in lipid-binding, and dynamin and N-WASP activation. We show that the broad lipid specificity of SNX9 is not because of independent and additive contributions by individual domains. Rather, the two domains appear to function in concert to confer lipid-binding and SNX9's membrane active properties. We also demonstrate that the two domains are differentially required for full SNX9 activity in N-WASP and dynamin regulation, and for localization of SNX9 to clathrin-coated pits and dorsal ruffles. In total, our results suggest that SNX9 can integrate signals from varied lipids through two domains to direct membrane remodeling events at multiple cellular locations.     

Live cell imaging of phosphoinositides with expressed inositide binding protein domains

Inositol lipids and calcium signaling has been inseparable twins during the 1980s when the molecular details of phospholipase C-mediated generation of inositol 1,4,5-trisphosphate (InsP3) and its Ca2+ mobilizing action were discovered. Since then, both the Ca2+ and inositol lipid signaling fields have hugely expanded and the tools allowing dissection of the finest details of their molecular organization also followed closely. Although phosphoinositides regulate many cell functions unrelated to Ca2+ signaling there are still many open questions even in the Ca2+ field that would benefit from single cell monitoring of PtdIns(4,5)P2 or InsP3 changes during agonist stimulation. This chapter is designed to provide practical guidance as well as some theoretical background on measurements of phosphoinositides in live cells using protein domain-GFP chimeras that could be also useful for people working on calcium signaling.     

Endophilin A1 regulates dendritic spine morphogenesis and stability through interaction with p140Cap

Dendritic spines are actin-rich membrane protrusions that are the major sites of excitatory synaptic input in the mammalian brain, and their morphological plasticity provides structural basis for learning and memory. Here we report that endophilin A1, with a well-established role in clathrin-mediated synaptic vesicle endocytosis at the presynaptic terminal, also localizes to dendritic spines and is required for spine morphogenesis, synapse formation and synaptic function. We identify p140Cap, a regulator of cytoskeleton reorganization, as a downstream effector of endophilin A1 and demonstrate that disruption of their interaction impairs spine formation and maturation. Moreover, we demonstrate that knockdown of endophilin A1 or p140Cap impairs spine stabilization and synaptic function. We further show that endophilin A1 regulates the distribution of p140Cap and its downstream effector, the F-actin-binding protein cortactin as well as F-actin enrichment in dendritic spines. Together, these results reveal a novel function of postsynaptic endophilin A1 in spine morphogenesis, stabilization and synaptic function through the regulation of p140Cap.     

Regulation of neuronal morphogenesis and synaptic function by Abl family kinases

Recent studies provide insights into the mechanisms by which Abelson non-receptor tyrosine kinases relay information from axon guidance and growth factor receptors to promote cytoskeletal rearrangements in developing neurons. Abelson non-receptor tyrosine kinases are also found in mature synapses, where their activities are required for optimal synaptic function.     

Binding of the PH and polybasic C-terminal domains of ARNO to phosphoinositides and to acidic lipids

The activity on ARF of the guanine nucleotide exchange factor ARNO depends on its membrane recruitment, induced by binding of its PH domain to phosphoinositides. A polycationic C-terminal extension to the PH domain might also contribute to its specific binding to phosphatidylinositol 4,5-bisphosphate [(4,5)PIP2] and to phosphatidylinositol 3,4,5-trisphosphate [(3,4,5)PIP3], and to ionic binding to other acidic lipids. We have analyzed in vitro the relative contributions to phospholipid binding of the PH domain and C-terminal extension by cosedimentation of "PH+C domain" and "nominal PH domain" protein constructs including or not including the polycationic C-terminus, with sucrose-loaded unilamellar vesicles made of equal proportions of the neutral lipids phosphatidylcholine and phosphatidylethanolamine, and supplemented or not with 30% acidic phosphatidylserine (PS) and 2% of various phosphoinositides. Binding was measured as a function of the vesicle concentration and of the medium ionic strength. Both proteins bound with higher affinity to (3,4,5)PIP3 than to (4,5)PIP2, the selectivity for (3,4,5)PIP3 being highest for the nominal PH domain. We observed also a clear selectivity of (3,4,5)PIP3 over (4,5)PIP2 for stimulating the activity of ARNO on ARF with vesicles containing 10% PS and 1% PIP2 or PIP3. Our data suggest that the PH domain provides the specific phosphoinositide binding site and some unspecific ionic interaction with acidic PS, whereas the polybasic C domain contributes to binding mainly by unspecific ionic interactions vith PS. Phosphorylation by protein kinase C of a serine in the C domain reduces the ionic affinity of the PH+C domain for PS, but does not affect the phosphoinositide specificity.     

Non-canonical interaction of phosphoinositides with pleckstrin homology domains of Tiam1 and ArhGAP9

Pleckstrin homology (PH) domains are phosphoinositide (PI)-binding modules that target proteins to membrane surfaces. Here we define a family of PH domain proteins, including Tiam1 and ArhGAP9, that demonstrates specificity for PI(4,5)P(2), as well as for PI(3,4,5)P(3) and PI(3,4)P(2), the products of PI 3-kinase. These PH domain family members utilize a non-canonical phosphoinositide binding pocket related to that employed by beta-spectrin. Crystal structures of the PH domain of ArhGAP9 in complex with the headgroups of Ins(1,3,4)P(3), Ins(1,4,5)P(3), and Ins(1,3,5)P(3) reveal how two adjacent phosphate positions in PI(3,4)P(2), PI(4,5)P(2), and PI(3,4,5)P(3) are accommodated through flipped conformations of the bound phospholipid. We validate the non-canonical site of phosphoinositide interaction by showing that binding pocket mutations, which disrupt phosphoinositide binding in vitro, also disrupt membrane localization of Tiam1 in cells. We posit that the diversity in PI interaction modes displayed by PH domains contributes to their versatility of use in biological systems.     

AMPA receptor trafficking pathways and links to dendritic spine morphogenesis

Alterations in synaptic strength are proposed to underlie learning and memory, and two major mechanisms utilised by neurons to bring about such changes involve regulating the number of AMPARs found at the synaptic plasma membrane, and altering the size and/or shape of the specialised postsynaptic compartment, the dendritic spine. It is now well-established that control of receptor number is brought about by precise modulation of vesicle trafficking, although the details of this crucial process are still far from clear. Regulation of spine size involves dynamic alterations in the spine actin cytoskeleton, but recent studies suggest that trafficking events may also play a role. In this review, I will summarise some recent findings about AMPAR trafficking pathways, and highlight the idea that membrane trafficking events not only regulate the complement of AMPARs at the synaptic plasma membrane, but also contribute to spine morphogenesis, either by regulating the plasma membrane content of spines, or as a result of changes in AMPAR trafficking.     

Application of phosphoinositide-binding domains for the detection and quantification of specific phosphoinositides

In mammals, seven phosphoinositides are known to play crucial roles as signaling molecules in a variety of cellular processes. Their synthesis and degradation are thought to be strictly controlled by metabolic enzymes such as phosphoinositide kinases and phosphatases, and their aberrant activities cause diseases. Thus, there is great interest in convenient and high-throughput measurement of such activities for the screening of drugs that enhance or block them. To date, radioactive labeling and colorimetric detection of released inorganic phosphates are mainly used to measure phosphoinositide kinase and phosphatase activities, respectively. Here, we describe a novel method for detecting and quantifying individual phosphoinositides via phosphoinositide-binding domains that exhibit high specificity and affinity toward this lipid. Enzyme-linked immunosorbent assay wells are modified with alkyl chains (C16), which enables more uniform and quantitative immobilization of phosphoinositide-containing liposomes onto the well surfaces. Phosphoinositides, as the substrate or the product, are detected by pleckstrin homology domains that specifically bind to each phosphoinositide. By this method, phosphoinositide contents are measured with higher sensitivities than those by conventional methods. More importantly, both phosphoinositide kinase and phosphatase activities can be measured for purified enzymes and crude cellular lysates. This assay is easy, sensitive, and quantitative and thus may have a variety of applications in the development of diagnostic tests or the screening of therapeutic treatments for diseases such as cancer and diabetes which may be caused by abnormal phosphoinositide metabolism.     

Keratinocyte TLR2 and TLR7 contribute to chronic itch through pruritic cytokines and chemokines in mice

Although neuronal Toll-like receptors (TLRs) (e.g., TLR2, TLR3, and TLR7) have been implicated in itch sensation, the roles of keratinocyte TLRs in chronic itch are elusive. Herein, we evaluated the roles of keratinocyte TLR2 and TLR7 in chronic itch under dry skin and psoriasis conditions, which was induced by either acetone-ether-water treatment or 5% imiquimod cream in mice, respectively. We found that TLR2 and TLR7 signaling were significantly upregulated in dry skin and psoriatic skin in mice. Chronic itch and epidermal hyperplasia induced by dry skin or psoriasis were comparably reduced in TLR2 and TLR7 knockout mice. In the dry skin model, the enhanced messenger RNA (mRNA) expression levels of pruritic CXCL1/2, IL-31, IL-33, ST2, IL-6, IL-17A, TNF-α, and IFN-γ were inhibited in TLR2^−/− mice, while CXCL2, IL-31, and IL-6 were inhibited in TLR7^−/− mice. In psoriasis model, the enhanced mRNA expression levels of pruritic CXCL1/2, IL-31, IL-33, ST2, IL-6, and TNF-α were inhibited in TLR2^−/− mice, while CXCL1/2, IL-31, IL-33, ST2, IL-6, IL-17A, and TNF-α were inhibited in TLR7^−/− mice. Incubation with Staphylococcus aureus (S. aureus) peptidoglycan (PGN-SA) (a TLR2 agonist), imiquimod (a TLR7 agonist), and miR142-3p (a putative TLR7 agonist) were sufficient to upregulate the expression of pruritic cytokines or chemokines in cultured keratinocyte HaCaT cells. Finally, pharmacological blockade of C-X-C Motif Chemokine Receptor 1/2 and high mobility group box protein 1 dose-dependently attenuated acute and chronic itch in mice. Together, these results indicate that keratinocyte TLR2 and TLR7 signaling pathways are distinctly involved in the pathogenesis of chronic itch.     

Second transmembrane domains of ENaC subunits contribute to ion permeation and selectivity

Epithelial sodium channels (ENaC) are composed of three structurally related subunits (alpha, beta, and gamma). Each subunit has two transmembrane domains termed M1 and M2, and residues conferring cation selectivity have been shown to reside in a pore region immediately preceding the M2 domains of the three subunits. Negatively charged residues are interspersed within the M2 domains, and substitution of individual acidic residues within human alpha-ENaC with arginine essentially eliminated channel activity in oocytes, suggesting that these residues have a role in ion permeation. We examined the roles of M2 residues in contributing to the permeation pore by individually mutating residues within the M2 domain of mouse alphaENaC to cysteine and systematically characterizing functional properties of mutant channels expressed in Xenopus oocytes by two-electrode voltage clamp. The introduction of cysteine residues at selected sites, including negatively charged residues (alphaGlu(595), alphaGlu(598), and alphaAsp(602)) led to a significant reduction of expressed amiloride-sensitive Na(+) currents. Two mutations (alphaE595C and alphaD602C) resulted in K(+)-permeable channels whereas multiple mutations altered Li(+)/Na(+) current ratios. Channels containing alphaD602K or alphaD602A also conducted K(+) whereas more conservative mutations (alphaD602E and alphaD602N) retained wild type selectivity. Cysteine substitution at the site equivalent to alphaAsp(602) within beta mENaC (betaD544C) did not alter either Li(+)/Na(+) or K(+)/Na(+) current ratios, although mutation of the equivalent site within gamma mENaC (gammaD562C) significantly increased the Li(+)/Na(+) current ratio. Mutants containing introduced cysteine residues at alphaGlu(595), alphaGlu(598), alphaAsp(602), or alphaThr(607) did not respond to externally applied sulfhydryl reagent with significant changes in macroscopic currents. Our results suggest that some residues within the M2 domain of alphaENaC contribute to the channel's conduction pore and that, in addition to the pore region, selected sites within M2 (alphaGlu(595) and alphaAsp(602)) may have a role in conferring ion selectivity.     

Juxtamembrane protein segments that contribute to recruitment of cholesterol into domains

We investigated the properties of several peptides with sequences related to LWYIK, a segment found in the gp41 protein of HIV and believed to play a role in sequestering this protein to a cholesterol-rich domain in the membrane. This segment fulfills the requirements to be classified as a CRAC motif that has been suggested to predict those proteins that will partition into cholesterol-rich regions of the membrane. All of the peptides were studied with the terminal amino and carboxyl groups blocked, i.e., as N-acetyl-peptide-amides. Effects of cholesterol on the intensity of W emission generally parallel DSC evidence of sequestration of cholesterol. Modeling studies indicate that all of these peptides tend to partition with their mass center at the membrane interface at the level of the hydroxyl of cholesterol. Interaction with cholesterol is dual: van der Waals interactions between mainly hydrophobic surfaces and electrostatic stabilization of the cholesterol OH group. Thus, both experiments and modeling studies indicate that the preference of CRAC motifs for cholesterol-rich domains might be related to a membrane interfacial preference of the motif, to a capacity to wrap and block the cholesterol polar OH group by H-bond interactions, and to a capacity for peptide aromatic side chains to stack with cholesterol. These results were supported by studies of single mutations in the gp41 protein of HIV-1, in which L(679) is replaced with I. Despite the similarity of the properties of these amino acid residues, this single substitution resulted in a marked attenuation of the ability of JC53-BL HeLa-based HIV-1 indicator cells to form syncytia.     

Coordination of tooth morphogenesis and neuronal development through tissue interactions: Lessons from mouse models

In addition to being an advantageous model to investigate general molecular mechanisms of organ formation, the tooth is a distinct target organ for peripheral nerve innervation. These nerves are required for the function and protection of the teeth and, as shown in fish, also for their regeneration. This review focuses on recent findings of the local tissue interactions and molecular signaling mechanisms that regulate the early nerve arrival and patterning of mouse mandibular molar tooth sensory innervation.     

Allosteric interactions between the myristate- and ATP-site of the Abl kinase

Abl kinase inhibitors targeting the ATP binding pocket are currently employed as potent anti-leukemogenic agents but drug resistance has become a significant clinical limitation. Recently, a compound that binds to the myristate pocket of Abl (GNF-5) was shown to act cooperatively with nilotinib, an ATP-competitive inhibitor to target the recalcitrant “T315I” gatekeeper mutant of Bcr-Abl. To uncover an explanation for how drug binding at a distance from the kinase active site could lead to inhibition and how inhibitors could combine their effects, hydrogen exchange mass spectrometry (HX MS) was employed to monitor conformational effects in the presence of both dasatinib, a clinically approved ATP-site inhibitor, and GNF-5. While dasatinib binding to wild type Abl clearly influenced Abl conformation, no binding was detected between dasatinib and T315I. GNF-5, however, elicited the same conformational changes in both wild type and T315I, including changes to dynamics within the ATP site located approximately 25 Å from the site of GNF-5 interaction. Simultaneous binding of dasatinib and GNF-5 to T315I caused conformational and/or dynamics changes in Abl such that effects of dasatinib on T315I were the same as when it bound to wild type Abl. These results provide strong biophysical evidence that allosteric interactions play a role in Abl kinase downregulation and that targeting sites outside the ATP binding site can provide an important pharmacological tool to overcome mutations that cause resistance to ATP-competitive inhibitors.     

The periplasmic domains of Escherichia coli HflKC oligomerize through right-handed coiled-coil interactions

The periplasmic domains of the Escherichia coli HflK and HflC were coexpressed and purified. The two polypeptides copurified in a 1:1 ratio, as determined by quantitative amino acid analysis. Circular dichroism studies showed the complex to have substantial helical/coiled-coil content that melted with midpoints in the range of 26-29 degrees C depending upon the concentration, implying a reversible oligomerization. The average molecular weight of the soluble HflKC determined by sedimentation equilibrium ultracentrifugation using a single-species model varied with rotor speed, providing further evidence of concentration-dependent oligomerization. The data were well-fit by models that specified a protomer to n-mer oligomerization, with the heterodimeric HflKC as the protomer and values of n between 7 and 10. Multiple-sequence alignments of both HflK and HflC revealed regions near the C termini to contain 11-residue hendecad repeats, indicative of right-handed coiled coils, with characteristic small residues in the a, f, h, and j positions. To test the importance of the small size of these positions, two residues in the HflC domain, Ala-262 in a f position and Gly-268 in an a position, were mutated to isoleucine. The HflKC:A262I mutant complex showed lower helicity than the wild type, and its melting was less concentration-dependent. During purification of HflKC:G268I, the mutated HflC subunit precipitated, leaving a preparation of the pure peripheral HflK domain. This polypeptide behaved as a monomer in sedimentation equilibrium experiments and showed low helicity, implying that the protein conformation is largely dependent upon heteromeric subunit interactions. These results demonstrate the importance of right-handed coiled-coil interactions in the oligomerization of HflKC, and a model entailing the formation of a right-handed helical barrel is proposed.     

Non-charged amino acids from three different domains contribute to link agonist binding to channel gating in alpha7 nicotinic acetylcholine receptors

Binding of agonists to nicotinic acetylcholine receptors results in channel opening. Previously, we have shown that several charged residues at three different domains of the alpha7 nicotinic receptor are involved in coupling binding and gating, probably through a network of electrostatic interactions. This network, however, could also be integrated by other residues. To test this hypothesis, non-charged amino acids were mutated and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Asn47 and Gln48 (loop 2), Ile130, Trp134, and Gln140 (loop 7), and Thr264 (M2-M3 linker) showed poor or null functional responses, despite significant membrane expression. By contrast, mutants F137A and S265A exhibited a gain of function effect. In all cases, changes in dose-response relationships were small, EC(50) values being between threefold smaller and fivefold larger, arguing against large modifications of agonist binding. Peak currents decayed at the same rate in all receptors except two, excluding large effects on desensitization. Thus, the observed changes could be mostly caused by alterations of the gating characteristics. Moreover, analysis of double mutants showed an interconnection between some residues in these domains, especially Gln48 with Ile130, suggesting a potential coupling between agonist binding and channel gating through these amino acids.